1. Field of the Invention
The present invention relates generally to the fields of optical imaging. More particularly, it concerns apparatus and methods for combining fluorescence and reflectance spectroscopy for the imaging of samples, including both in situ and ex situ imagining of body tissues.
2. Description of Related Art
Cancer is one of the leading causes of death in the United States and in the world. In the United States alone, deaths from cancer are estimated to number 560,000 in 1997 (American Cancer Society Online. Cancer Facts and Figures). Currently, diagnosis and treatment of cancer follow histopathologic evaluation of directed biopsies. However, the tissue removal necessitated by these techniques not only may alter the progression of the disease (Robbins and Kumar, 1984) but is also very costly. Improving the capability for in situ monitoring of disease progression could greatly enhance the ability to detect and treat cancer and precancer (Kelloff et al., 1992).
A growing number of clinical studies have demonstrated that fluorescence spectroscopy may be used to distinguish normal and abnormal human tissues in vivo in the skin, head and neck, genito-urinary tract, gastro-intestinal tract, breast, and brain. It is well known that fluorescence intensity and lineshape are a function of both the excitation and emission wavelength in samples containing multiple chromophores, such as human tissue. A complete characterization of the fluorescence properties of an unknown sample requires measurement of a fluorescence excitation emission matrix, in which the fluorescence intensity is recorded as a function of both excitation and emission wavelength. The field of analytical chemistry has exploited the fluorescence properties of different compounds to identify and quantify them in mixtures.
Most clinical studies reported to date have measured fluorescence emission spectra at only a small number of excitation wavelengths (typically one to three) due to clinical requirements imposed on the size, speed and sensitivity of instrumentation. The choice of excitation wavelength has been based on factors which vary from study to study, but include laser availability and predictions of chromophores thought to be present in normal and abnormal tissues and measurements of fluorescence excitation emission matrices (EEM) of normal and abnormal tissues in vitro. While in vitro measurements of tissue EEMs are feasible using commercially available scanning fluorimeters, several studies have demonstrated that the optical properties of tissue change significantly when tissue is examined in vitro due in part to interruption of the blood supply, oxidation and small size of biopsies. Thus, in vitro studies to select excitation wavelengths are of limited value.
Several recent studies have suggested that differences in optical properties, assessed using diffuse reflectance spectroscopy, may be used to discriminate normal and abnormal human tissues in vivo in the urinary bladder and the skin. Furthermore, measuring both fluorescence and diffuse reflectance spectra may provide additional information of diagnostic value.
A system capable of measuring spatially resolved reflectance spectra and fluorescence excitation emission matrices in vivo would remove limitations of many previous studies, potentially enabling prediction of excitation wavelengths that provide greatest discrimination of normal and abnormal tissues, as well as a better understanding of the relative diagnostic ability of changes in absorption, scattering and fluorescence properties of tissue. Although fiber optic systems to record fluorescence EEMs and reflectance spectra at a single spatial location have been reported, such systems have measured data from only a single spatial location, and have thus not been able to perform spatially resolved spectroscopy. Additionally, previous systems have not been well-adapted for in-vivo studies of various tissues.
In one respect, the invention is an apparatus for performing fluorescence and spatially resolved reflectance spectroscopy on a sample, and it includes a light source, a monochromator, a reflectance illumination fiber, a fluorescence excitation fiber, an imaging spectrograph, a fluorescence collection fiber, a reflectance collection fiber, and a detector. The monochromator is in optical communication with the light source. The reflectance illumination fiber is in optical communication with the light source. The fluorescence excitation fiber is in optical communication with the monochromator. The fluorescence collection fiber is in optical communication with the imaging spectrograph. The reflectance collection fiber is in optical communication with the imaging spectrograph and is in spaced relation with the reflectance illumination fiber. The detector is in optical communication with the imaging spectrograph.
In other aspects, the light source may include a Xe arc lamp. The monochromator may include a double monochromator. The detector comprises a thermo-electrically cooled CCD camera. The fluorescence excitation fiber and the fluorescence collection fiber may be integral. One or more of the fibers may be positioned flush with the sample. The apparatus may also include a spacer positioned between one or more of the fibers and the sample. The reflectance illumination fiber, the fluorescence excitation fiber, the fluorescence collection fiber, and the reflectance collection fiber may define a fiber optic probe. The probe may be configured to be positioned within a trocar. The probe may include a center section and an outer section, and the fluorescence excitation fiber and the fluorescence collection fiber may be positioned in the center section, and the reflectance illumination fiber and the reflectance collection fiber may be positioned in the outer section. The apparatus may include a plurality of fluorescence excitation and collection fibers arranged in a circular bundle. The apparatus may include a plurality of reflectance collection fibers defining a plurality of collection positions. The plurality of collection positions may be spaced between about 0 and about 10 millimeters from the reflectance illumination fiber. The reflectance collection fiber may define a collection position at about 180 degrees relative to the reflectance illumination fiber. The reflectance collection fiber may define a collection position at about 90 degrees relative to the reflectance illumination fiber. The reflectance collection fiber may define a collection position at about 45 degrees relative to the reflectance illumination fiber. The apparatus may include one or more fibers in optical communication with the light source and configured to illuminate the sample during operation of the apparatus. The apparatus may include a plurality of fluorescence excitation fibers arranged in one or more rows adjacent the monochromator. The apparatus may include a plurality of fluorescence excitation fibers and a plurality of reflectance collection fibers arranged in a single row adjacent the imaging spectrograph. The apparatus may include one or more unconnected fibers interspersed with the plurality of fluorescence excitation fibers and the plurality of reflectance collection fibers. The apparatus may include a fiber connected from the light source to the imaging spectrograph to monitor spectral output of the light source. The apparatus may include a controller coupled to the detector.
In another respect, the invention is an apparatus for measuring fluorescence and spatially resolved reflectance spectra of a sample. The apparatus includes a light source, a monochromator, a fiber optic probe, an imaging spectrograph, and a detector. The monochromator is in optical communication with the light source. The fiber optic probe is in optical communication with the light source and with the monochromator. The probe includes a plurality of fluorescence excitation and collection fibers in spaced relation and a plurality of reflectance collection fibers in spaced relation with a reflectance illumination fiber. The imaging spectrograph is in optical communication with the plurality of fluorescence collection fibers and with the plurality of reflectance collection fibers. The detector is in optical communication with the imaging spectrograph.
In other aspects, the plurality of reflectance collection fibers and the reflectance illumination fiber may be positioned concentrically about the plurality of fluorescence excitation and collection fibers. At least one of the plurality of reflectance collection fibers may define a collection position at about 180 degrees relative to the reflectance illumination fiber. At least one of the plurality of reflectance collection fibers may define a collection position at about 90 degrees relative to the reflectance illumination fiber. At least one of the plurality of reflectance collection fibers may define a collection position at about 45 degrees relative to the reflectance illumination fiber. The plurality of collection positions may be spaced between about 0 and about 10 millimeters from the reflectance illumination fiber. The probe may include between twenty-one and forty-six optical fibers.
In another respect, the invention is a method for combined fluorescence and spatially resolved reflectance spectroscopy of a sample. The method includes directing radiation to the sample with a fluorescence excitation fiber, collecting radiation from the sample with a fluorescence collection fiber, directing the radiation from the sample to an imaging spectrograph and a detector, illuminating the sample with a reflectance illumination fiber, collecting reflected light from the sample with a reflectance collection fiber in spaced relation with the reflectance illumination fiber, and directing the reflected light from the sample to an imaging spectrograph and a detector.
In other aspects, the step of collecting reflected light may include collecting reflected light from a plurality of collection positions with a plurality of reflectance collection fibers. The step of collecting reflected light may include collecting reflected light from the sample with a reflectance collection fiber defining a collection position at about 180 degrees relative to the reflectance illumination fiber. The step of collecting reflected light may include collecting reflected light from the sample with a reflectance collection fiber defining a collection position at about 90 degrees relative to the reflectance illumination fiber. The step of collecting reflected light may include collecting reflected light from the sample with a reflectance collection fiber defining a collection position at about 45 degrees relative to the reflectance illumination fiber. The sample may include ovarian, head and neck, or cervical tissue. The method may also include analyzing spectral data from the detector to characterize the sample. The step of analyzing may include pre-processing the data and reducing a dimension of the data using principal component analysis. The step of analyzing may also include selecting one or more diagnostic principal components of the data and forming one or more algorithms. The step of analyzing may also include forming one or more composite algorithms. The step of analyzing may also include evaluating at least on of the algorithms using a cross-validation technique.
In another respect, the invention is a method for combined fluorescence and spatially resolved reflectance spectroscopy of a sample. The method includes directing radiation to the sample with a fluorescence excitation fiber, collecting radiation from the sample with a fluorescence collection fiber, directing the radiation from the sample to an imaging spectrograph and a detector, illuminating the sample with a reflectance illumination fiber, collecting reflected light at a plurality of collection positions from the sample with a plurality of reflectance collection fibers arranged in spaced relation, directing the reflected light from the sample to an imaging spectrograph and a detector to produce spectral data, pre-processing the data, and reducing a dimension of the data using principal component analysis.
The method may also include selecting one or more diagnostic principal components of the data and forming one or more algorithms. The method may also include forming one or more composite algorithms. The method may also include evaluating at least one of the algorithms using a cross-validation technique.
In another respect, the invention is a method for analyzing spectroscopy data to define an optimized reduced data set. The method includes pre-processing the spectroscopy data, reducing a dimension of the spectroscopy data using principal component analysis, and selecting one or more diagnostic principal components of the spectroscopy data.
In other aspects, the spectroscopy data may include combined fluorescence and spatially resolved reflectance spectroscopy data. The step of pre-processing may include normalization of the spectroscopy data. The step of pre-processing may include mean scaling the spectroscopy data. The step of pre-processing may include calculating one or more derivatives on the spectroscopy data. The method may also include eliminating redundant data from the spectroscopy data. The method may also include forming one or more algorithms and evaluating at least one of the algorithms using a cross validation technique. The method may also include forming one or more composite algorithms.
Applications for the methods and apparatus described herein are vast and include, but are not limited to, analysis and detection of disease including cancers and pre-cancers (such as cervical, head and neck, colon, lung, esophageal, ovarian) and atherosclerosis. Applications also include industry, including, but not limited to, the semiconductor industry.